GABA reuptake

(R)-N-[4,4-Bis(3-methyl-2-thienyl)but-3-en-1-yl]nipecotic acid (Tiagabine/NO 328) has previously been shown to be a potent anticonvulsant in both mice and rats. Here, we report that NO 328 is a potent inhibitor of gamma-[3H]aminobutyric acid [( 3H]GABA) uptake in a rat forebrain synaptosomal preparation (IC50 = 67 nM) and in primary cultures of neurons and astrocytes. Inhibition of [3H]GABA uptake by NO 328 is apparently of a mixed type when NO 328 is preincubated before [3H]GABA uptake; the inhibition is apparently competitive without preincubation. NO 328 itself is not a substrate for the GABA uptake carrier, but Tiagabine/NO 328 is a selective inhibitor of [3H]GABA uptake. Binding to benzodiazepine receptors, histamine H1 receptors, and 5-hydroxytryptamine1A receptors was inhibited by NO 328 at 5-30 microM, whereas several other receptors and uptake sites were unaffected. [3H]NO 328 showed saturable and reversible binding to rat brain membranes in the presence of NaCl. The specific binding of [3H]NO 328 was inhibited by known inhibitors of [3H]GABA uptake; GABA and the cyclic amino acid GABA uptake inhibitors were, however, less potent than expected. This indicates that the binding site is not identical to, but rather overlapping with, the GABA recognition site of the uptake carrier. The affinity constant for binding of [3H]NO 328 is 18 nM, and the Bmax is 669 pmol/g of original rat forebrain tissue. The regional distribution of NaCl-dependent [3H]NO 328 binding followed that of synaptosomal [3H]GABA uptake [1].

---

We studied in vitro the effects of Tiagabine on genomic DNA of cortical rat astrocytes. To evaluate DNA damage, we used a relatively simple technique called Single Cell Gel Electrophoresis or Comet assay. Tiagabine was dissolved in culture medium and added at concentration of 1, 10, 20 and 50 μg/ml on 12-day old cultured astrocytes. In presence of 1 and 10 μg/ml of Tiagabine, no DNA damage was observed after 48 h of treatment. A moderate DNA damage was instead observed for cells exposed to 20 μg/ml of antiepileptic drug. Finally, DNA fragmentation was more evident after treatment with 50 μg/ml of Tiagabine. We conclude that Tiagabine, at the usual recommended doses, does not appear to influence negatively the cortical rat astrocytes, inducing DNA fragmentation only at very high concentrations.[4]

γ-aminobutyric acid (GABA) has been implicated in the pathophysiology of anxiety disorders, including panic. Tiagabine, a selective GABA reuptake inhibitor (SGRI), has been shown to reduce symptoms of anxiety. This pilot study evaluated the efficacy and safety of tiagabine in patients with panic disorder. Male and female outpatients aged 18-64 years with a DSM-IV diagnosis of severe to moderately severe panic disorder (with or without agoraphobia) received open-label tiagabine 2-20 mg/day for 10 weeks. Outcome assessments included the Sheehan Panic Disorder Scale (SPS), Panic Disorder Severity Scale (PDSS), Bandelow Panic and Agoraphobia Scale (PAS), Hamilton Rating Scale for Anxiety (HAM-A), 21-point Clinician Global Improvement Scale (CGI-21), 21-point Patient Global Improvement (PGI-21) and the Sheehan Disability Scale (SDS). Scores were recorded at baseline and weekly intervals thereafter. Adverse events were monitored throughout the study. Of the 28 patients who enrolled in the study, 23 had one post-baseline visit and were available for LOCF outcome analysis. Although statistically significant reductions from baseline were observed for all of the outcome measures, the percentage improvements on individual scales were only in the 25-32% range which is not clinically significant. Tiagabine was generally well tolerated; the most common adverse events were nausea, dizziness and headaches. Only one patient discontinued tiagabine due to adverse events. These findings suggest that administration of tiagabine may be of little benefit in patients with panic disorder.[2]

---

Effect of Tiagabine on GABA uptake in gray and white structures [3]
To identify the type of GABA transporter involved in white matter high-affinity GABA uptake we studied the sensitivity of GABA uptake to Tiagabine. Tiagabine, a selective inhibitor of the GAT-1 GABA transporter with a Ki of ∼0.1 μM (Thomsen et al., 1997) inhibited high-affinity GABA uptake in both temporal cortex and white structures (Fig. 1B). At 0.1 μM tiagabine high-affinity GABA uptake was inhibited ∼50% in proteoliposomes made from temporal cortex. In the white structures 0.1 μM tiagabine caused 60–68% inhibition, which was significantly greater than the inhibition seen in temporal cortex (Fig. 1B). Even at 0.33 μM tiagabine high-affinity GABA uptake was slightly more inhibited in corpus callosum, pyramidal tracts and white matter underlying the occipital cortex than in temporal cortex (Fig. 1B).

Astrocyte primary cultures were prepared from 1±2 dayold Wistar rats, decapitated and the cells obtained as described by Cardile et al. The astrocytes were cultivated in Dulbecco's modi®ed Eagle's medium /F12 medium containing 10% fetal calf serum, 1 mM L-glutamine and antibiotics and incubated at 378C in humidi®ed 5% CO2/ 95% atmosphere. At 12 days, the different cell plates were separately considered and processed either as untreated control or Tiagabine treated astrocytes. To assess the astroglial nature of the cells, the test for glial ®brillary acidic protein was performed before the beginning of the treatments. In the present study, only data from cultures where immunostaining for glial ®brillary acidic protein showed that 90±95% of cells were astrocytes have been considered. The Comet assay was applied according to Singh et al, with little modi®cation. Brie¯y, monolayer astrocytes (untreated control and differently Tiagabine treated for 48 h) were washed twice with phosphate-buffered saline (PBS) at pH 7.4, suspended in 2 ml of PBS by scraping the dishes with a rubber policeman and centrifuged at 800 g for 15 min. The pellet was re-suspended in a small volume of PBS, and an aliquot of the cells was mixed with 0.4% trypan blue solution and counted. The remaining cells were considered for Comet assay. Microscope slides were cleaned with 100% methanol, air dried, and rinsed with a solution of standard melting point agarose (NMA) at 1% (w/v) and solidi®ed. Ten microliters of cell suspension (0.8±1 £ 105 cells) were mixed with 75 ml of 0.5% low melting point agarose (LMA) and spotted on slides. A third layer of 85 ml LMA was then added. The `minigels' were immersed for 1 h at 48C in ice cold lysis solution (N-laurosil-sarcosine 1%, NaCl 2.5 M, Na2EDTA 10 mM, Triton X-100 1%, DMSO 10%, (pH 10)), denatured in a high pH buffer (NaOH 300 mM, Na2EDTA 1 mM) for 20 min, and run in the same buffer for 25 min at 25 V in an ice bath and under semi-dark conditions. At the end of the electrophoresis, the slides were washed gently three times in a neutralization buffer (Tris±HCl 0.4 M, (pH 7.5)), stained with 100 ml of ethidium bromide (2 mg/ml) for 10 min and covered with an 18 £ 18 mm cover slip; the dye in excess was absorbed. The slides were either scored immediately, using a Leitz ¯uorescence microscope interfaced with a computer. [4]

High-affinity uptake of [3H]GABA into proteoliposomes and fresh homogenates [3]
Plasma membrane transporters for GABA were reconstituted in proteoliposomes according to the method of Danbolt et al. (1990) as described (Trotti et al., 1995, Hassel et al., 2003), starting from 10% homogenates (w/v) in sucrose, 0.32 M. These proteoliposomes have an internal buffer of KCl, 140 mM. Uptake of [3H]GABA, 0.5 μM, final specific activity 5.6 mCi/μmol, was performed in triplicates in the presence of NaCl, 150 mM, and valinomycin, 1 μM, for 15 min at 30 °C; the sequestered radioactivity was trapped on filter paper and quantified by scintillation counting. Blanks were run in duplicates for each structure and were obtained by keeping the samples on ice and adding the sodium ionophore nigericin, 1 μM. Uptake was linear with time for at least 20 min. Previously, uptake by proteoliposomes made from frozen brain tissue has been shown to be the same as for fresh brain homogenates when [3H]glutamate was the transported compound (Hassel et al., 2003), suggesting that freezing does not affect transport activities.
The sensitivity of high-affinity GABA uptake to Tiagabine was investigated in temporal cortex and white structures. Tiagabine, a selective inhibitor of the GAT-1 GABA transporter, with a Ki of 0.1 μM (Thomsen et al., 1997), was added during incubation at 0.1 or 0.33 μM.

Absorption, Distribution and Excretion
Tigaben is almost completely absorbed (>95%). After oral administration, approximately 2% of tigaben is excreted unchanged, with 25% and 63% of the remaining dose excreted in urine and feces, respectively, primarily as metabolites. 109 mL/min [Healthy Subjects] Tigaben is rapidly absorbed, reaching peak plasma concentrations approximately 45 minutes after oral administration on an empty stomach. Tigaben is almost completely absorbed (>95%), with an absolute oral bioavailability of approximately 90%. High-fat meals may decrease the absorption rate of tigaben (mean time to peak is prolonged to 2.5 hours, and mean peak concentration is reduced by approximately 40%), but do not affect its extent of absorption (area under the curve). The pharmacokinetics of tigaben are linear over a single dose range of 2 to 24 mg. Steady state is reached within 2 days after multiple administrations.
Tigaben binds to human plasma proteins in 96% of cases, primarily serum albumin and α1-acid glycoprotein, at concentrations ranging from 10 ng/mL to 10,000 ng/mL. While the relationship between Tiagabinen plasma concentrations and clinical efficacy is currently unclear, trough concentrations observed at daily doses of 30 to 56 mg ranged from <1 ng/mL to 234 ng/mL in controlled clinical trials.
A diurnal rhythm effect was observed in the pharmacokinetics of Tiagabinen. The mean steady-state Cmin value was 40% lower in the evening than in the morning. The steady-state AUC value was also 15% lower after evening administration of Tiagabinen compared to morning administration.
For more complete data on the absorption, distribution, and excretion of Tiagabinens (9 in total), please visit the HSDB record page.
Metabolism/Metabolites

Tigaben is likely primarily metabolized by the 3A subfamily of hepatocyte pigment P450.
Although the metabolism of Tiagabine is not fully elucidated, in vivo and in vitro studies have identified at least two metabolic pathways in humans: 1) epoxidation of thiophene to 5-oxo-Tiagabine; 2) glucuronidation. The 5-oxo-Tiagabine metabolite does not participate in the pharmacological activity of Tiagabine.
Based on in vitro data, Tiagabine is likely primarily metabolized by the hepatic cytochrome P450 3A subfamily (CYP 3A), but the possibility that CYP 1A2, CYP 2D6, or CYP 2C19 may also be involved in Tiagabine metabolism cannot be ruled out.
Tiagabine is likely primarily metabolized by the hepatic cytochrome P450 3A subfamily.
Elimination pathway: After oral administration of Tiagabine, approximately 2% is excreted unchanged, and the remaining 25% and 63% are excreted in urine and feces, respectively, primarily as metabolites.
Half-life: 7-9 hours

---

Biological half-life
7-9 hours
……In healthy subjects, the mean elimination half-life of Tiagabine is 7 to 9 hours. Compared with patients with hepatic enzyme-induced epilepsy who did not experience it, the elimination half-life in patients with hepatic enzyme-induced epilepsy was shortened by 50% to 65%.
Its half-life is approximately 8 hours, but when used in combination with hepatic enzyme-inducing drugs such as phenobarbital, phenytoin, or carbamazepine, the half-life can be shortened by 2 to 3 hours.

Toxicity Summary
While the exact mechanism of action of tiagabine in humans is unclear, it appears to be a selective GABA reuptake inhibitor. Hepatotoxicity
Limited data exist regarding tiagabine hepatotoxicity. In clinical trials, tiagabine treatment was not associated with elevated serum transaminases or an increased incidence of hepatotoxicity. No case reports of liver injury caused by tiagabine have been published, and its use has not been found to be associated with hypersensitivity syndromes or autoimmune diseases. However, its overall use is limited. Probability Score: E (Unlikely a cause of clinically apparent liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation Infants should be monitored for lethargy, weight gain, and developmental milestones, especially in younger, exclusively breastfed infants and when using anticonvulsants or psychotropic drugs in combination. Due to very limited experience with tiagabine use during lactation, alternative medications may be preferred, especially in breastfed newborns or preterm infants.
◉ Effects on Breastfed Infants
One mother breastfed her infant while taking tiagabine 24 mg/day, then reduced to 20 mg/day.
Ten newborns aged 4 to 23 days were breastfed while their mothers were taking levetiracetam 1000 to 3000 mg daily, with no adverse reactions reported. One mother was concurrently taking tiagabine 30 mg/day, clobazian 45 mg/day, and oxcarbazepine 600 mg/day.
◉ Effects on Lactation and Breast Milk
As of the revision date, no relevant published information was found.

---

Protein Binding
96%Interaction
/Concomitant use of tiagabine with alcohol or central nervous system depressants may exacerbate central nervous system depression.
In patients taking carbamazepine, phenobarbital, phenytoin, or primidone, tiagabine clearance is increased by 60%.
Tigaben causes a slight decrease (approximately 10%) in the steady-state concentration of valproic acid; in vitro studies have shown that valproic acid can reduce the protein binding rate of Tiagabinen from 96.3% to 94.8%, resulting in an increase in the concentration of free Tiagabinen by approximately 40%; the clinical significance of this finding is unclear.
Concomitant use of cimetidine (800 mg/day) in patients taking Tiagabinen long-term has no effect on the pharmacokinetics of Tiagabinen.
For more complete data on drug interactions of Tiagabinen (13 items in total), please visit the HSDB record page.

[1]. (R)-N-[4,4-bis(3-methyl-2-thienyl)but-3-en-1-yl]nipecotic acid binds with high affinity to the brain gamma-aminobutyric acid uptake carrier. J Neurochem. 1990 Feb;54(2):639-47.
[2]. An open-label study of tiagabine in panic disorder. Psychopharmacol Bull, 2007. 40(3): p. 32-40.
[3]. High-affinity GABA uptake and GABA-metabolizing enzymes in pig forebrain white matter: a quantitative study. Neurochem Int, 2007. 50(2): p. 365-70.
[4]. Tiagabine treatment and DNA damage in rat astrocytes: an in vitro study by comet assay. Neurosci Lett. 2001 Jun 22;306(1-2):17-20.

Tiagabine hydrochloride is the hydrochloride salt formed by the reaction of equimolar amounts of Tiagabine with hydrogen chloride. It is a GABA reuptake inhibitor used to treat epilepsy. It has a dual effect of anticonvulsant and GABA reuptake inhibition. It contains Tiagabine(1+) ions. Tiagabine hydrochloride is the hydrochloride form of Tiagabine, a nipoise derivative with anticonvulsant activity. Tiagabine hydrochloride inhibits γ-aminobutyric acid (GABA) transporter type 1 (GAT1), which is primarily located at the presynaptic terminals of neurons, thereby preventing the reuptake of GABA by the presynaptic terminals. Therefore, this increases the level of available GABA in the synaptic cleft, thus prolonging its inhibitory effect. Tiagabine is effective against electroconvulsive seizures as well as limbic system and generalized tonic-clonic seizures. Tiagabine is a nipoise derivative used as both a GABA reuptake inhibitor and an anticonvulsant. It is used to treat epilepsy, especially refractory partial seizures. See also: Tiagabine (containing the active moiety). Tiagabine is a piperidine monocarboxylic acid, a derivative of (R)-nipoic acid, in which the hydrogen atom bonded to the nitrogen atom is replaced by 1,1-bis(3-methyl-2-thienyl)but-1-en-4-yl. It is a GABA reuptake inhibitor, commonly used in the form of hydrochloride to treat epilepsy. It has a dual role as both a GABA reuptake inhibitor and an anticonvulsant. It is a piperidine monocarboxylic acid, β-amino acid, thiophene compound, and tertiary amine compound. Its function is related to (R)-nipoic acid. It is the conjugate base of Tiagabine (1+). Tiagabine is an anticonvulsant. It is also used to treat panic disorder, like some other anticonvulsants. Although the exact mechanism by which Tiagabine works in the human body is not fully understood, it appears to be a selective GABA reuptake inhibitor. Tiagabine is an antiepileptic drug. The physiological effects of Tiagabine are achieved by reducing disordered electrical activity in the central nervous system.
Tiagabine is a unique anticonvulsant primarily used as adjunctive therapy in the treatment of partial-onset seizures in adults or children. Treatment with tiagabine does not cause elevated serum transaminases, and there are no reports of clinically significant liver damage caused by tiagabine; even if it occurs, it is extremely rare.
Tiagabine is an anticonvulsant. It is also used to treat panic disorder, like some other anticonvulsants. Although the exact mechanism of action of tiagabine in humans is unclear, it appears to be a selective GABA reuptake inhibitor.
Tiagabine is a nipoise derivative with GABA reuptake inhibitor and anticonvulsant activity. It is used to treat epilepsy, especially refractory partial-onset seizures.
See also: Tiagabine hydrochloride (in salt form). Tiagabine hydrochloride monohydrate (its active ingredient).
Drug Indications

For the treatment of partial-onset seizures
FDA Label
Mechanism of Action

Although the exact mechanism of action of tiagabine in humans is unclear, it appears to be a selective GABA reuptake inhibitor. Although the exact mechanism of action of Tiagabine is not fully understood, this drug enhances GABA-mediated inhibitory neurotransmission. Tiagabine increases GABA levels in the extracellular spaces of the globus pallidus, ventral globus pallidus, and substantia nigra, suggesting a GABA-mediated anticonvulsant mechanism (i.e., inhibition of nerve impulse transmission leading to seizures). Tiagabine inhibits the reuptake of GABA by presynaptic neurons and glial cells and increases the amount of GABA available for postsynaptic receptor binding. The drug does not stimulate GABA release and, at concentrations inhibiting GABA uptake, is inactive at other receptor binding and uptake sites. Tiagabine selectively blocks presynaptic GABA uptake by reversibly and saturatingly binding to recognition sites associated with GABA transporters on neuronal and glial cell membranes. In vitro binding studies have shown that thiagaben has no significant inhibitory effect on the uptake of dopamine, norepinephrine, serotonin, glutamate, or choline, and its binding to dopamine D1 or D2 receptors, cholinergic muscarinic receptors, and serotonergic type 1A, 2, or 3 receptors (5HT1A, 5HT2, or 5HT3, respectively) is also insignificant. It also binds to α1- or α2-adrenergic receptors; β1- or β2-adrenergic receptors; histamine H2 or H3 receptors; adenosine A1 or A2 receptors; opioid μ or κ1 receptors; glutamate N-methyl-D-aspartate (NMDA) receptors; or GABAA receptors. Furthermore, thiagaben has very low or no affinity for sodium or calcium channels. Thiagabe binds to histamine H1 receptors, 5-HT1B receptors, benzodiazepine receptors, and chloride channel receptors at concentrations 20-400 times higher than those inhibiting GABA uptake.

---

Therapeutic Use
Thiagabe is indicated as adjunctive therapy to other antiepileptic drugs for the treatment of partial seizures in adults and children aged 12 years and older. /US Product Label Content/

---

Drug Warnings
While thiagbene can reduce the frequency of seizures in patients with epilepsy, its use has been associated with ambivalent seizures in patients without a history of epilepsy.
Post-marketing reports have shown that thiagbene use has been associated with new-onset epilepsy and status epilepticus in patients without a history of epilepsy. Dosage may be a significant triggering factor for seizures, although seizures have been reported in patients taking as little as 4 mg of thiagbene daily. In most cases, patients are taking other medications (antidepressants, antipsychotics, stimulants, anesthetics) that are thought to lower the seizure threshold. Some seizures occur before or after dose increases, even when the previous dose was stable. The current label dosage recommendations for tiagabine in treating epilepsy are based on use in patients aged 12 years and older with partial seizures, most of whom are taking enzyme-inducible antiepileptic drugs (AEDs; such as carbamazepine, phenytoin sodium, primidone, and phenobarbital), which reduce tiagabine's plasma concentration by inducing its metabolism. When tiagabine is used alone without combination with an AED, plasma concentrations are approximately twice those observed in studies on which the current dosage recommendations are based. The safety and efficacy of tiagabine have not been established, and its indication is limited to adjunctive therapy for partial seizures in adults and children aged 12 years and older. For more complete data on drug warnings for tiagabine (19 in total), please visit the HSDB record page.

---

Pharmacodynamics
Tiagabine is primarily used as an anticonvulsant for adjunctive treatment of epilepsy. The exact mechanism by which Tiagabine exerts its antiepileptic effect is unclear, but it is believed to be related to its ability to enhance the activity of γ-aminobutyric acid (GABA, the main inhibitory neurotransmitter in the central nervous system). Tiagabine binds to recognition sites associated with GABA uptake carriers. It is speculated that Tiagabine blocks the uptake of GABA by presynaptic neurons through this action, thereby making more GABA available to bind to receptors on the postsynaptic cell surface. Based on our current and previous findings, we can conclude that Tiagabine does not appear to have a negative effect on rat cortical astrocytes at commonly recommended doses, and only induces DNA fragmentation at very high concentrations. However, since astrocytes are more resistant to oxidative stress than neurons, the possibility that Tiagabine may also induce neuronal DNA fragmentation at lower concentrations cannot be ruled out. [4]

C20H26CLNO2S2

412.003

411.109

C, 58.31; H, 6.36; Cl, 8.60; N, 3.40; O, 7.77; S, 15.56

145821-59-6

Tiagabine;115103-54-3;Tiagabine hydrochloride hydrate;145821-57-4

91274

White to off-white solid powder

568ºC at 760 mmHg

>192oC dec.

297.3ºC

9.71E-14mmHg at 25°C

5.784

2

5

6

26

474

1

CC1=C(SC=C1)C(=CCCN2CCC[C@H](C2)C(=O)O)C3=C(C=CS3)C.Cl

YUKARLAABCGMCN-PKLMIRHRSA-N

InChI=1S/C20H25NO2S2.ClH/c1-14-7-11-24-18(14)17(19-15(2)8-12-25-19)6-4-10-21-9-3-5-16(13-21)20(22)23;/h6-8,11-12,16H,3-5,9-10,13H2,1-2H3,(H,22,23);1H/t16-;/m1./s1

(R)-1-(4,4-bis(3-methylthiophen-2-yl)but-3-en-1-yl)piperidine-3-carboxylic acid hydrochloride

NNC-05-0328; NO-05-0328; NO329; Tiagabine hydrochloride; 145821-59-6; TIAGABINE HCl; Abbott 70569.HCl; Abbott-70569.1; NNC-05-0328; NO-05-0328; ABBOTT-70569.HCL; ...NNC-050328; NO 329; trade name Gabitril

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~100 mg/mL (~242.71 mM)
DMSO : ≥ 53 mg/mL (~128.64 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.05 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (5.05 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (5.05 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)